A procedure for measuring total serum thyroxine by radioactive techniques was first developed by Ekins, as disclosed in Clin. Chim. Acta 5:453 (1960). In that procedure, which was termed "Saturation Analysis," extracted thyroxine (T4) was added to standard plasma solution containing radioactive iodine (.sup.131 I) labeled T4. Electrophoretic procedures were used to measure the shift of labeled T4 from globulin to albumin with the amount displaced being a measure of patient T4.
Murphy and Pattee (J. Clin. Endocrinol. 24:187 (1964)) describe a method of total serum thyroxine determination ("Competitive Protein Binding Analysis") similar to that of Ekins but technically simpler and more rapid. Bound T4 is released from the serum proteins by denaturation and precipitation with ethanol. This procedure leaves approximately 80 percent of the T4 in the alcoholic supernatant. After the supernatant is evaporated to dryness, a buffered reagent containing labeled T4, thyroxine binding globulin (TBG) and barbital is added to the extract. During a period of incubation, the extracted T4 competes with radioactive T4 for binding sites on the TBG. Thereafter, the solution is passed through a column of crosslinked dextran gel, or is exposed to anion-exchange resin beads, and the free thyroxine is bound to the gel or anion-exchange resin. The radioactive-labeled T4 bound to the gel, or to the resin beads, is proportional to the amount of T4 present in the original sample.
A more complete description of the prior art is set forth in U.S. Pat. Nos. 3,414,383 and 3,799,740, and in the references cited therein. The use of polystyrene anion-exchange resin beads to bind free T4 (both labeled and unlabeled) following an equilibrating step in which a solution of free extracted T4 from a sample of patient's serum competes with a known quantity of radioactive-labeled T4 bound to saturated TBG, is now common practice in clinical laboratories. Nevertheless, such beads do present problems and inconveniences both in production and in laboratory use. For example, even though such beads are relatively large (usually 35 mesh or larger), the weighing and handling of such beads are difficult whether performed in the laboratory or in a manufacturing facility. Keeping the beads together throughout a test may itself be a problem; should a single bead be lost during the performance of a test, the difference would significantly alter the results.
Furthermore, the use of conventional resin beads involves manipulative steps which are not only time consuming but, at least in some instances, increase the risks of bead loss. Thus, it has been generally considered necessary to carry out the competitive-binding equilibration step in advance of bead introduction, allowing such equilibration to take place in what amounts to an incubation step (usually at room temperature) over an interval of 5 to 15 minutes. Thereafter, the beads are introduced, intimately mixed with the fluid and again incubated for a similar interval. Two incubation periods are regarded as necessary; should the first incubation be omitted, reproducibility of test results is affected, apparently because the presence of large porous resin beads somehow interferes with or at least delays the development of equilibrium conditions during the competitive-binding step.
Following the equilibration step, the supernatant and the resin beads are separated and the radioactivity of one or the other (or both) is measured by means of a suitable gamma counter. However, before the actual measurement of radioactivity is taken, standard procedure requires that the resin beads be washed to remove residual TBG-bound thyroxine, again for the purpose of achieving reproducible results. Such a washing step is obviously time consuming, requires manipulative effort, and presents further possibilities for error. For example, should one or more beads be lost during the washing step, the test results might be seriously affected where the procedure involves measuring radioactivity from the resin; should less than all of the wash liquid be added to the supernatant, the accuracy of the results would again be jeopardized in a procedure where radioactive readings are taken from the supernatant.
While it has been suggested that the handling and measurement problems might be reduced if somehow the anion-exchange resin beads could be pressed into the form of a pellet or tablet, no such product is known to have become available in the past. The resilient porous nature of the polystyrene beads makes it virtually impossible to compress them to a stable compacted condition in which they will remain after the compressive force is removed. Binders (such as starch) are known but no binder systems for anion-exchange resin beads have been suggested which would permit tableting and at the same time would not interfere with proper functioning of the beads during a test procedure. Therefore, while certain of the advantages of tableting have been recognized, those advantages have not been realized in the past for anion-exchange resins, nor has there been any suggestion as to just how such advantages might be realized.